AB0036 HIGH THROUGHPUT siRNA EFFICACY SCREENING USING FLUORESCENCE SYSTEM
Authors: M. Deniz, L. Hakimi Pour, A. S. Thorup, F. Dell’accio
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Osteoarthritis (OA) is characterized by loss of articular cartilage, bone remodelling and pain. It is a leading cause of disability in the elderly population. Currently, there are no disease modifying drugs to stop progression of OA. One bottleneck in clinical translation is that the cartilage extracellular matrix is avascular and impenetrable for large macromolecules. siRNA induces highly specific gene silencing and can be used for targets that are "undruggable". A novel therapeutic target that has been identified for treatment of OA include using siRNA to silence receptor tyrosine kinase–like orphan receptor 2 (ROR2) which offers great potential in improving pain outcomes and structural integrity in OA mice (Thorup et al., 2020). However, efficacy of "naked" siRNA is limited as it is instantly targeted for degradation by nucleases. To improve utility of siRNA as a therapeutic modality, chemical modifications can be introduced to the siRNA backbone, and it can be coupled with carrier molecules. <h3>Objectives</h3> To identify RNA backbone modifications to achieve long-term silencing in joint tissues following intra-articular injection. <h3>Methods</h3> Cos7 cells transduced with a lentivirus encoding EGFP and one specific clone was selected and expanded for downstream experiments (Eldridge <i>et al.</i>, 2016). Transfections were performed using JetPrime. Modified siRNA was generated by Integrated DNA Technologies. <h3>Results</h3> As a screening system, we optimized a microtitre plate system coupled with live fluorescent detection to measure GFP expression in cell monolayers. The fluorescence system uses a 96-well plate and allows for repeat measurements over time, without the need to harvest samples at each timepoint. Using this system, we compared 5 different modifications were compared. The best modification allowed to silence GFP in GFP-expressing cells for at least 10 days. The best modification was coupled with atelocollagen and administered via intra-articular injections into mice expressing GFP ubiquitously. After 3 weeks, GFP was efficiently silenced in the full thickness of the cartilage. <h3>Conclusion</h3> Chemical modifications of the RNA backbone and coupling with atelocollagen affords stable gene silencing in cartilage for at least 3 weeks. This technology enables fast, dose-dependent loss of function experiments in animal models of osteoarthritis. This methodology could be developed in the future to therapeutically target pathogenic molecules that are out of reach of traditional biologics such as transcription factors. <h3>References</h3> [1]Eldridge, S. <i>et al.</i> (2016) 'Agrin mediates chondrocyte homeostasis and requires both LRP4 and α-dystroglycan to enhance cartilage formation in vitro and in vivo', <i>Annals of the Rheumatic Diseases</i>. doi: 10.1136/annrheumdis-2015-207316. [2]Thorup, A. S. <i>et al.</i> (2020) 'ROR2 blockade as a therapy for osteoarthritis', <i>Science Translational Medicine</i>. doi: 10.1126/SCITRANSLMED.AAX3063. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.